Gla domains as targeting agents

ABSTRACT

The disclosure relates to the recombinant Gla domain proteins and their use targeting phosphatidylserine (PtdS) moieties on the surface of cells, particularly those expressing elevated levels of PtdS, such as cells undergoing apoptosis.

This application claims benefit of priority to U.S. ProvisionalApplication Ser. No. 61/791,537 filed Mar. 15, 2013, and U.S.Provisional Application Ser. No. 61/787,753, filed Mar. 15, 2013, theentire contents of which are hereby incorporated by reference.

BACKGROUND

1. Field

This disclosure relates to the targeting of phosphatidylserine (PtdS) oncell membranes using Gla domain peptides and polypeptides. The use ofthese peptides and polypeptides as therapeutic agents is disclosed.

2. Related Art

Phosphatidlyserine (PtdS) is a negatively charged phospholipid componentusually localized to the inner-leaflet (the cytoplasmic side) of thecell membrane. However, PtdS can be transported by scramblase (a memberof the flippase family) from the inner-leaflet to the outer-leaflet andexposed on the cell surface. With very few exceptions, this activeexternalization of PtdS is a response to cellular damage (van den Eijndeet al., 2001; Erwig and Henson, 2008). For example, tissue injurysignals platelets, leukocytes, and endothelial cells to rapidly andreversibly redistribute PtdS which leads to the promotion of coagulationand complement activation on cell surfaces. Similarly, apoptotic signalsresult in the externalization of PtdS however in a more gradual andsustained manner. This external PtdS provides a key recognition markerthat enables macrophages to ingest dying cells from surrounding tissuewhile suppressing a full and detrimental immune response (Erwig andHenson, 2008). This removal process is essential for tissue homeostasisand in a “healthy” environment it is extremely efficient. In fact,despite the loss of >10⁹ cells per day, the histological detection ofapoptotic cells is a rare event in normal tissues (Elltiot andRavichandran, 2010; Elltiot et al., 2009). However, there is evidencethat in many pathological conditions the process of apoptotic cellremoval is overwhelmed, delayed or absent (Elltiot and Ravichandran,2010; Lahorte et al., 2004). For example several oncology studiessuggest that a high apoptotic index is associated with higher gradetumors, increased rate of metastasis and a poor prognosis for thepatient (Naresh et al., 2001; Loose et al., 2007; Kurihara et al., 2008;Kietselaer et al., 2002). These studies, and others like them, suggestthat apoptosis and external PtdS expression can be a powerful marker ofdisease (Elltiot and Ravichandran, 2010).

There are several proteins with a high affinity for anionic phospholipidsurfaces with Annexin-V being the most widely utilized as a PtdStargeting probe (Lahorte et al., 2004). With a high affinity for PtdScontaining vesicles (K_(d)=0.5-7 nM) and a molecular weight (37 kDa)that falls below the threshold for kidney filtration (approx. 60 kDa)Annexin-V has shown promise in the clinic as an apoptosis-probe (Lin etal., 2010; Tait and Gibson, 1992). Moreover, it has been utilized for awide range of indications including those in oncology, neurology andcardiology (Lahorte et al., 2004; Boersma et al., 2005; Blankenberg,2009; Reutelingsperger et al., 2002). The use of biologic probes whichtarget PtdS cell-surface expression has been shown both in vitro and invivo. While their utility in the clinic is promising, they have, for themost part, not yet been exploited.

SUMMARY

Thus, in accordance with the present disclosure, there is provided amethod of targeting cell membrane phosphatidylserine (PtdS) comprising(a) providing an isolated polypeptide comprising agamma-carboxyglutamic-acid (Gla) domain and lacking a protease orhormone-binding domain; and (b) contacting the peptide with a cellsurface, wherein the polypeptide binds to PtdS on the cell membrane. Thecell membrane may be a cardiac cell membrane, a neuronal cell membrane,an endothelial cell membrane, a virus-infected cell membrane, anapoptotic cell membrane, a platelet membrane, a plasma membrane-derivedveriscle (PMV) or a cancer cell membrane. The polypeptide may furthercomprise an EGF binding domain, a Kringle domain, and/or an aromaticamino acid stack domain. The Gla domain may be from Factor II, FactorVII, Factor IX, Factor X, protein S or protein C. The polypeptide mayfurther comprises a detectable label, such as a fluorescent label, achemiluminescent label, a radiolabel, an enzyme, a dye or a ligand.

The polypeptide may be 300 residues or less, 200 residues or less, or100 residues or less, including ragnes of 100-200 and 100-300 residues.The polypeptide may comprise 5-15 Gla residues, 9-13 Gla residues,including 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 Gla residues. Thepolypeptide may comprise more than 13 Gla residues, but less than 30%total Gla residues. The polypeptide may be between about 4.5 and 30 kDin size. The polypeptide may comprise at least one disulfide bond, or2-5 disulfide bonds. The polypeptide may comprise a protein S Gladomain. The polypeptide may comprise a protein S Gla domain, a protein SEGF domain, a prothrombin Gla domain, a prothrombin Gla domain plusprothrombin Kringle domain, a protein Z Gla domain, a protein Z Gladomain plus prothrombin Kringle domain, a Factor VII Gla domain, or aFactor VII Gla domain plus prothrombin Kringle domain. The polypeptidemay further comprise an antibody Fc region. Any of the foregoing maycontain conservative substitutions of the native sequences for theforegoing proteins, and/or exhibit a percentage homology to the nativedomains set forth.

In another embodiment, there is provided a method of treating cancer ina subject comprising administering to the subject an isolatedpolypeptide comprising a gamma-carboxyglutamic-acid (Gla) domain andlacking a protease or hormone-binding domain. The cancer may be breastcancer, brain cancer, stomach cancer, lung cancer, prostate cancer,ovarian cancer, testicular cancer, colon cancer, skin cancer, rectalcancer, cervical cancer, uterine cancer, liver cancer, pancreaticcancer, head & neck cancer or esophageal cancer. The method may furthercomprise treating the subject with a second cancer therapy, such as animmunotherapy, a radiotherapy, a chemotherapy, a toxin therapy, acytokine therapy or a hormone therapy.

Yet another embodiment includes a method of treating an autoimmunedisease in a subject comprising administering to the subject an isolatedpolypeptide comprising a gamma-carboxyglutamic-acid (Gla) domain andlacking a protease or hormone-binding domain. The autoimmune disease maybe spondyloarthropathy, ankylosing spondylitis, psoriatic arthritis,reactive arthritis, enteropathic arthritis, ulcerative colitis, Crohn'sdisease, irritable bowel disease, inflammatory bowel disease, rheumatoidarthritis, juvenile rheumatoid arthritis, familial Mediterranean fever,amyotrophic lateral sclerosis, Sjogren's syndrome, early arthritis,viral arthritis, multiple sclerosis, systemic lupus erythematosus,psoriasis, vasculitis, Wegener's granulomatosis, Addison's disease,alopecia, antiphospholipid syndrome, Behcet's disease, celiac disease,chronic fatigue syndrome, ulcerative colitis, type I diabetes,fibromyalgia, autoimmune gastritis, Goodpasture syndrome, Graves'disease, idiopathic thrombocytopenic purpura (ITP), myasthenia gravis,pemphigus vulgaris, primary biliary cirrhosis, rheumatic fever,sarcoidosis, scleroderma, vitiligo, vasculitis, small vessel vasculitis,hepatitis, primary biliary cirrhosis, sarcoidosis, scleroderma, graftversus host disease (acute and chronic), aplastic anemia, or cyclicneutropenia. The method may further comprise treating the subject with asecond autoimmune disease therapy, such as prednisone, methylprednisone,Venipred, Celestone, hydrocortisone, triamcinoclone, AristonpanIntra-Articular injection, Methapred, Rayos oral, betamethasone, oretanercept.

In still another embodiment, there is provided a method of treating aviral disease in a subject comprising administering to the subject anisolated polypeptide comprising a gamma-carboxyglutamic-acid (Gla)domain and lacking a protease or hormone-binding domain. The viraldisease may be influenza, human immunodeficiency virus, dengue virus,West Nile virus, smallpox virus, respiratory syncytial virus, Koreanhemorrhagic fever virus, chickenpox, varicella zoster virus, herpessimplex virus 1 or 2, Epstein-Barr virus, Marburg virus, hantavirus,yellow fever virus, hepatitis A, B, C or E, Ebola virus, human papillomavirus, rhinovirus, Coxsackie virus, polio virus, measles virus, rubellavirus, rabies virus, Newcastle disease virus, rotavirus, HTLV-1 and -2.The method may further comprise treating the subject with a secondanti-viral therapy, such as Abacavir, Aciclovir, Acyclovir, Adefovir,Amantadine, Amprenavir, Ampligen, Arbidol, Atazanavir, Atripla,Boceprevirertet, Cidofovir, Combivir, Darunavir, Delavirdine,Didanosine, Docosanol, Edoxudine, Efavirenz, Emtricitabine, Enfuvirtide,Entecavir, Entry inhibitors, Famciclovir, Fomivirsen, Fosamprenavir,Foscarnet, Fosfonet, Ganciclovir, Ibacitabine, Imunovir, Idoxuridine,Imiquimod, Indinavir, Inosine, Integrase inhibitor, Interferon type III,Interferon type II, Interferon type I, Interferon, Lamivudine,Lopinavir, Loviride, Maraviroc, Moroxydine, Methisazone, Nelfinavir,Nevirapine, Nexavir, Nucleoside analogues, Oseltamivir, Peginterferonalfa-2a, Penciclovir, Peramivir, Pleconaril, Podophyllotoxin, Proteaseinhibitor, Raltegravir, Reverse transcriptase inhibitor, Ribavirin,Rimantadine, Ritonavir, Pyramidine, Saquinavir, Stavudine, Synergisticenhancer (antiretroviral), Tea tree oil, Telaprevir, Tenofovir,Tenofovir disoproxil, Tipranavir, Trifluridine, Trizivir, Tromantadine,Truvada, Valaciclovir, Valganciclovir, Vicriviroc, Vidarabine,Viramidine, Zalcitabine, Zanamivir or Zidovudine.

In still a further embodiment, there is provided a method of treating ahypercoagulation disorder in a subject comprising administering to thesubject an isolated polypeptide comprising a gamma-carboxyglutamic-acid(Gla) domain and lacking a protease or hormone-binding domain. Themethod may further comprise treating the subject with one or moreadditional anti-coagulants.

Also provided are methods of modulating clotting in a subject comprisingadministering to the subject an isolated polypeptide comprising agamma-carboxyglutamic-acid (Gla) domain and lacking a protease orhormone-binding domain. The method may further comprise administering tothe subject a clotting factor.

Yet another embodiment comprises treating sepsis in a subject comprisingadministering to the subject an isolated polypeptide comprising agamma-carboxyglutamic-acid (Gla) domain and lacking a protease orhormone-binding domain. A still further embodiment includes a method oftreating vaso-occlusive crisis in a sickle cell subject comprisingadministering to the subject an isolated polypeptide comprising agamma-carboxyglutamic-acid (Gla) domain and lacking a protease orhormone-binding domain.

Finally, there is provided a method treating a disorder characterized bypathologic expression of phosphatidylserine on the surface of a cellcomprising administering to the subject an isolated polypeptidecomprising a gamma-carboxyglutamic-acid (Gla) domain and lacking aprotease or hormone-binding domain.

It is contemplated that any method or composition described herein canbe implemented with respect to any other method or composition describedherein.

Other objects, features and advantages of the present disclosure willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating specific embodiments of the disclosure, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the disclosure will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE FIGURES

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentdisclosure. The disclosure may be better understood by reference to oneor more of these drawings in combination with the detailed.

FIG. 1—Construction of a panel of Gla and Gla-EGF/Kringle domainproteins.

FIG. 2—Testing of Gla domain protein constructs for expression.Transient transfection into 293 cells using 293cellFectin. 10% gels withreduced samples, 23.3 μl of media loaded.

FIG. 3—Testing of Gla domain protein constructs for expression.Transient transfection in BHK21 cells. 10% gels with reduced samples, 20μl ( 1/100 total cell pellet) loaded.

FIG. 4—Changing signal sequence alter secretion. Transient transfectionin BHK21 cells. 10% gels with reduced samples, 13.3 μl loaded.

FIG. 5—Protein S Gla+EGF sequence.

FIG. 6—Purification of Protein S Gla+EGF. F1-F4 are columnchromatography fractions. 10% gels, non-reducing conditions.

FIG. 7—Apoptosis Assays for Protein S Gla+EGF. Top and bottom panelsrepresent identical duplicate procedures except that amounts of ProteinS Gla+EGF was reduced, and the amount of anti-His domain antibody wasreduced.

FIG. 8—Apoptosis Assays for Protein S Gla+EGF. Top and bottom panelsrepresent identical duplicate procedures except for amounts of Annexin Vused, which are double in the bottom panels.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

Like annexin, gamma-carboxyglutamic-acid (Gla)-domain proteins such asFactors II, VII, IX, X, protein C, and protein S bind anionic membranes.In fact, the Gla-domain has been used as a model for a small moleculethat was rationally designed to be an apoptosis-specific probe (Cohen etal., 2009). Here, the inventors propose the utilization of the membranetargeting portions of these Gla-domain proteins as a novel class ofbiological probes specific for apoptosis and disease. The use of thesenaturally-occurring and targeted proteins may lead to enhancedspecificity relative to current probes with the added advantage of asmaller size (<30 kDa). Even in larger embodiments, which would includeEGF and/or Kringle domains, these proteins can still be smaller thanAnnexin V (37 kDa), and potentially as small as <5 kDa. These biologicprobes can target PtdS cell-surface expression both in vitro and invivo. Thus, it is possible to develop an apoptosis/disease targetingprobe that is superior to Annexin V in affinity, specificity and sizewith the added potential for use as a therapeutic. These and otheraspects of the disclosure are described in greater detail below.

Whenever appropriate, terms used in the singular will also include theplural and vice versa. In the event that any definition set forth belowconflicts with the usage of that word in any other document, includingany document incorporated herein by reference, the definition set forthbelow shall always control for purposes of interpreting thisspecification and its associated claims unless a contrary meaning isclearly intended (for example in the document where the term isoriginally used). The use of “or” means “and/or” unless statedotherwise. The use of “a” herein means “one or more” unless statedotherwise or where the use of “one or more” is clearly inappropriate.The use of “comprise,” “comprises,” “comprising,” “include,” “includes,”and “including” are interchangeable and are not limiting. For example,the term “including” shall mean “including, but not limited to.” Theword “about” means plus or minus 5% of the stated number.

An “isolated peptide or polypeptide,” as used herein, is intended torefer to a peptide or polypeptide which is substantially free of otherbiological molecules, including peptides or polypeptides having distinctsequences. In some embodiments, the isolated peptide or polypeptide isat least about 75%, about 80%, about 90%, about 95%, about 97%, about99%, about 99.9% or about 100% pure by dry weight. In some embodiments,purity can be measured by a method such as column chromatography,polyacrylamide gel electrophoresis, or HPLC analysis.

As used herein, “conservative substitutions” refers to modifications ofa polypeptide that involve the substitution of one or more amino acidsfor amino acids having similar biochemical properties that do not resultin loss of a biological or biochemical function of the polypeptide. A“conservative amino acid substitution” is one in which the amino acidresidue is replaced with an amino acid residue having a similar sidechain. Families of amino acid residues having similar side chains havebeen defined in the art. These families include amino acids with basicside chains (e.g., lysine, arginine, histidine), acidic side chains(e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g.,glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine),nonpolar side chains (e.g., alanine, valine, leucine, isoleucine,proline, phenylalanine, methionine, tryptophan), β-branched side chains(e.g., threonine, valine, isoleucine), and aromatic side chains (e.g.,tyrosine, phenylalanine, tryptophan, histidine). Antibodies of thepresent disclosure can have one or more conservative amino acidsubstitutions yet retain antigen binding activity.

For nucleic acids and polypeptides, the term “substantial homology”indicates that two nucleic acids or two polypeptides, or designatedsequences thereof, when optimally aligned and compared, are identical,with appropriate nucleotide or amino acid insertions or deletions, in atleast about 80% of the nucleotides or amino acids, usually at leastabout 85%, in some embodiments about 90%, 91%, 92%, 93%, 94%, or 95%, inat least one embodiment at least about 96%, 97%, 98%, 99%, 99.1%, 99.2%,99.3%, 99.4%, or 99.5% of the nucleotides or amino acids. Alternatively,substantial homology for nucleic acids exists when the segments willhybridize under selective hybridization conditions to the complement ofthe strand. Also included are nucleic acid sequences and polypeptidesequences having substantial homology to the specific nucleic acidsequences and amino acid sequences recited herein.

The percent identity between two sequences is a function of the numberof identical positions shared by the sequences (i.e., % homology=# ofidentical positions/total # of positions×100), taking into account thenumber of gaps, and the length of each gap, which need to be introducedfor optimal alignment of the two sequences. Comparison of sequences anddetermination of percent identity between two sequences can beaccomplished using a mathematical algorithm, such as without limitationthe AlignX™ module of VectorNTI™ (Invitrogen Corp., Carlsbad, Calif.).For AlignX™, the default parameters of multiple alignment are: gapopening penalty: 10; gap extension penalty: 0.05; gap separation penaltyrange: 8; % identity for alignment delay: 40. (further details atworld-wide-web atinvitrogen.com/site/us/en/home/LINNEA-nline-Guides/LINNEA-Communities/Vector-NTI-Community/Sequence-analysis-and-data-management-software-for-PCs/AlignX-Module-for-Vector-NTI-Advance.reg.us.html).

Another method for determining the best overall match between a querysequence (a sequence of the present disclosure) and a subject sequence,also referred to as a global sequence alignment, can be determined usingthe CLUSTALW computer program (Thompson et al., Nucleic Acids Res, 1994,2(22): 4673-4680), which is based on the algorithm of Higgins et al.,Computer Applications in the Biosciences (CABIOS), 1992, 8(2): 189-191).In a sequence alignment the query and subject sequences are both DNAsequences. The result of the global sequence alignment is in percentidentity. Parameters that can be used in a CLUSTALW alignment of DNAsequences to calculate percent identity via pairwise alignments are:Matrix=IUB, k-tuple=1, Number of Top Diagonals=5, Gap Penalty=3, GapOpen Penalty=10, Gap Extension Penalty=0.1. For multiple alignments, thefollowing CLUSTALW parameters can be used: Gap Opening Penalty=10, GapExtension Parameter=0.05; Gap Separation Penalty Range=8; % Identity forAlignment Delay=40.

The nucleic acids can be present in whole cells, in a cell lysate, or ina partially purified or substantially pure form. A nucleic acid is“isolated” or “rendered substantially pure” when purified away fromother cellular components with which it is normally associated in thenatural environment. To isolate a nucleic acid, standard techniques suchas the following can be used: alkaline/SDS treatment, CsCl banding,column chromatography, agarose gel electrophoresis and others well knownin the art.

I. PHOSPHATIDYLSERINE (PTDS)

A. Structure and Synthesis

Phosphatidylserine (abbreviated PtdS, Ptd-L-Ser or PS) is a phospholipidcomponent, usually kept on the inner-leaflet (the cytosolic side) ofcell membranes by an enzyme called flippase. When a cell undergoesapoptosis, phosphatidylserine is no longer restricted to the cytosolicpart of the membrane, but becomes exposed on the surface of the cell.The chemical formula of PtdS is C₁₃H₂₄NO₁₀P and has a molecular mass of385.304. The structure is shown below:

Phosphatidylserine is biosynthesized in bacteria by condensing the aminoacid serine with CDP (cytidine diphosphate)-activated phosphatidic acid.In mammals, phosphatidylserine is produced by base-exchange reactionswith phosphatidylcholine and phosphatidylethanolamine. Conversely,phosphatidylserine can also give rise to phosphatidylethanolamine andphosphatidylcholine, although in animals the pathway to generatephosphatidylcholine from phosphatidylserine only operates in the liver.

B. Function

Early studies of phosphatidylserine distilled the chemical from bovinebrain. Modern studies and commercially available products are made fromsoybeans, because of concerns about mad cow disease. The fatty acidsattached to the serine in the soy product are not identical to those inthe bovine product and is also impure. Preliminary studies in ratsindicate that the soy product is at least as potent as that of bovineorigin.

The U.S. FDA has given “qualified health claim” status tophosphatidylserine, stating that, “Consumption of phosphatidylserine mayreduce the risk of dementia in the elderly” and “Consumption ofphosphatidylserine may reduce the risk of cognitive dysfunction in theelderly.”

Phosphatidylserine has been demonstrated to speed up recovery, preventmuscle soreness, improve well-being, and might possess ergogenicproperties in athletes involved in cycling, weight training andendurance running Soy-PtdS, in a dose dependent manner (400 mg), hasbeen reported to be an effective supplement for combatingexercise-induced stress by blunting the exercise-induced increase incortisol levels. PtdS supplementation promotes a desirable hormonalbalance for athletes and might attenuate the physiological deteriorationthat accompanies overtraining and/or overstretching. In recent studies,PtdS has been shown to enhance mood in a cohort of young people duringmental stress and to improve accuracy during tee-off by increasing thestress resistance of golfers. First pilot studies indicate that PtdSsupplementation might be beneficial for children with attention-deficithyperactivity disorder.

Traditionally, PtdS supplements were derived from bovine cortex (BC-PS);however, due to the potential transfer of infectious diseases,soy-derived PS (S-PS) has been established as a potential safealternative. Soy-derived PS is Generally Recognized As Safe (GRAS) andis a safe nutritional supplement for older persons if taken up to adosage of 200 mg three times daily. Phosphatidylserine has been shown toreduce specific immune response in mice.

PtdS can be found in meat, but is most abundant in the brain and ininnards such as liver and kidney. Only small amounts of PS can be foundin dairy products or in vegetables, with the exception of white beans.

Annexin-A5 is a naturally-occurring protein with avid binding affinityfor PtdS. Labeled-annexin-A5 enables visualization of cells in theearly- to mid-apoptotic state in vitro or in vivo. Another PtdS bindingprotein is Mfge8. Technetium-labeled annexin-A5 enables distinctionbetween malignant and benign tumors whose pathology includes a high rateof cell division and apoptosis in malignant compared with a low rate ofapoptosis in benign tumors.

II. GLA DOMAIN PROTEINS

A. Gla Domains

The general structure for the Gla-domain proteins is that of a Gladomain followed by EGF domains and then a C terminal serine proteasedomain. The exceptions are prothrombin, which contains Kringle domainsin place of EGF domains, and protein S, which does not have a serineprotease domain but rather sex hormone-binding globulin-like (SHBG)domains (Hansson and Stenflo, 2005). The affinities of Gla-domainproteins to anionic membranes vary. Roughly, they fall into 3categories 1) high affinity binders with a Kd of 30-50 nM, 2)mid-affinity binders with a K_(d) of 100-200 nM and 3) low affinitybinders with a Kd of 1000-2000 nM. The high affinity Gla domain proteinshave been shown to bind anionic membranes with Protein S specificallydemonstrating binding to apoptotic cells via its interaction with PtdS(Webb et al., 2002). The low affinity Gla domain proteins use asecondary receptor to bind to the cell membrane. For example, FVIIutilizes Tissue Factor (TF). The Gla domain/1^(st) EGF domain isbelieved to constitute the high affinity TF binding domain of FVII.Importantly for this approach, there are many studies that have shown TFup-regulation on the surface of cancer cells including colorectalcancer, NSCL carcinoma, and breast cancer and these high TF levels havebeen associated with a poor prognosis (Yu et al., 2004). Although theaffinity for anionic membranes is relatively low for FVII, the additionof the high affinity TF interaction along with the documentedup-regulation of TF in cancer makes it a potentially interesting cancerspecific probe.

B. Gla Domain Containing Proteins

1. Factor II

Prothrombin, also known as coagulation factor II, is proteolyticallycleaved to form thrombin in the coagulation cascade, which ultimatelyresults in the stemming of blood loss. Thrombin in turn acts as a serineprotease that converts soluble fibrinogen into insoluble strands offibrin, as well as catalyzing many other coagulation-related reactions.It is primarily expressed in the liver.

The gene encoding prothrombin is located on chromosome 11 in the regionof the centromere. It is composed of 14 exons and contains 24 kilobasesof DNA. The gene encodes a signal region, a propeptide region, aglutamic acid domain, 2 Kringle regions, and a catalytic domain. Theenzyme gamma-glutamyl carboxylase, in the presence of vitamin K,converts the N-terminal glutamic acid residues to gamma-carboxyglutamicacid residues. These gamma-carboxyglutamic acid residues are necessaryfor the binding of prothrombin to phospholipids on platelet membranes.

Inherited factor II deficiency is an autosomal recessive disorder thatcan manifest as hypoprothrombinemia, a decrease in the overall synthesisof prothrombin, or as dysprothrombinemia, the synthesis of dysfunctionalprothrombin. Homozygous individuals are generally asymptomatic and havefunctional prothrombin levels of 2-25%. However, symptomatic individualsmay experience easy bruising, epistaxis, soft-tissue hemorrhage,excessive postoperative bleeding, and/or menorrhagia.

Prothrombin plays a role in a role in chronic urticaria, an autoimmunedisease, and various vascular disorders. Livedo vasculopathy isassociated with immunoglobulin (Ig)M antiphosphatidylserine-prothrombincomplex antibody. The presence of antiphosphatidylserine-prothrombincomplex antibodies and histopathological necrotizing vasculitis in theupper-to-middle dermis indicates cutaneous leukocytoclastic angiitisrather than cutaneous polyarteritis nodosa.

Aside from the prothrombin deficiencies, another disorder of prothrombinis the prothrombin 20210a mutation. A familial cause of venousthromboembolism, the prothrombin 20210a mutation results in increasedlevels of plasma prothrombin and a concurrent increased risk for thedevelopment of thrombosis. Although the exact mechanism of this disorderhas not been elucidated, the prothrombin 20210a mutation involves thesubstitution of an adenine for a guanine at position 20210 within the 3′untranslated region of the prothrombin gene. This mutation alters thepolyadenylation site of the gene and results in increased mRNAsynthesis, with a subsequent increase in protein expression.

2. Factor VII

Factor VII (formerly known as proconvertin) is one of the proteins thatcauses blood to clot in the coagulation cascade. The gene for factor VIIis located on chromosome 13 (13q34). It is an enzyme of the serineprotease class, and recombinant form of human factor VIIa (NovoSeven)has U.S. Food and Drug Administration approval for uncontrolled bleedingin hemophilia patients. It is sometimes used unlicensed in severeuncontrollable bleeding, although there have been safety concerns. ABiosimilar form of recombinant activated factor VII (AryoSeven) ismanufactured by AryoGen Biopharma.

The main role of factor VII (FVII) is to initiate the process ofcoagulation in conjunction with tissue factor (TF/factor III). Tissuefactor is found on the outside of blood vessels—normally not exposed tothe bloodstream. Upon vessel injury, tissue factor is exposed to theblood and circulating factor VII. Once bound to TF, FVII is activated toFVIIa by different proteases, among which are thrombin (factor Ha),factor Xa, IXa, XIIa, and the FVIIa-TF complex itself. The mostimportant substrates for FVIIa-TF are Factor X and Factor IX. Factor VIIhas been shown to interact with Tissue factor (TF).

The action of the factor is impeded by tissue factor pathway inhibitor(TFPI), which is released almost immediately after initiation ofcoagulation. Factor VII is vitamin K dependent; it is produced in theliver. Use of warfarin or similar anticoagulants decreases hepaticsynthesis of FVII.

Deficiency is rare (congenital proconvertin deficiency) and inheritsrecessively. Factor VII deficiency presents as a hemophilia-likebleeding disorder. It is treated with recombinant factor VIIa (NovoSevenor AryoSeven). Recombinant factor VIIa is also used for people withhemophilia (with Factor VIII or IX deficiency) who have developedinhibitors against replacement coagulation factor. It has also been usedin the setting of uncontrollable hemorrhage, but its role in thissetting is controversial with insufficient evidence to support its useoutside of clinical trials. The first report of its use in hemorrhagewas in an Israeli soldier with uncontrollable bleeding in 1999. Risks ofits use include an increase in arterial thrombosis.

3. Factor IX

Factor IX (or Christmas factor) is one of the serine proteases of thecoagulation system; it belongs to peptidase family S1. The gene forfactor IX is located on the X chromosome (Xq27.1-q27.2) and is thereforeX-linked recessive: mutations in this gene affect males much morefrequently than females. Deficiency of this protein causes hemophilia B.Factor IX is produced as a zymogen, an inactive precursor. It isprocessed to remove the signal peptide, glycosylated and then cleaved byfactor XIa (of the contact pathway) or factor VIIa (of the tissue factorpathway) to produce a two-chain form where the chains are linked by adisulfide bridge. When activated into factor IXa, in the presence ofCa²⁺, membrane phospholipids, and a Factor VIII cofactor, it hydrolysesone arginine-isoleucine bond in factor X to form factor Xa. Factor IX isinhibited by antithrombin.

Factors VII, IX, and X all play key roles in blood coagulation and alsoshare a common domain architecture. The factor IX protein is composed offour protein domains. These are the Gla domain, two tandem copies of theEGF domain and a C-terminal trypsin-like peptidase domain which carriesout the catalytic cleavage. The N-terminal EGF domain has been shown toat least in part be responsible for binding Tissue factor. Wilkinson etal. conclude that residues 88 to 109 of the second EGF domain mediatebinding to platelets and assembly of the Factor X activating complex.The structures of all four domains have been solved. A structure of thetwo EGF domains and trypsin like domain was determined for the pigprotein. The structure of the Gla domain, which is responsible forCa(II)-dependent phospholipid binding, was also determined by NMR.Several structures of “super active” mutants have been solved whichreveal the nature of Factor IX activation by other proteins in theclotting cascade.

Deficiency of factor IX causes Christmas disease (hemophilia B). Over100 mutations of factor IX have been described; some cause no symptoms,but many lead to a significant bleeding disorder. Recombinant factor IXis used to treat Christmas disease, and is commercially available asBeneFIX. Some rare mutations of factor IX result in elevated clottingactivity, and can result in clotting diseases, such as deep veinthrombosis.

4. Factor X

Factor X (Stuart-Prower factor; prothrombinase) is an enzyme of thecoagulation cascade. The human factor X gene is located on thethirteenth chromosome (13q34). It is a serine endopeptidase (proteasegroup S1). Factor X is synthesized in the liver and requires vitamin Kfor its synthesis. Factor X is activated into factor Xa by both factorIX (with its cofactor, factor VIII in a complex known as intrinsic Xase)and factor VII with its cofactor, tissue factor (a complex known asextrinsic Xase). The half life of factor X is 40-45 hours. It istherefore the first member of the final common pathway or thrombinpathway. It acts by cleaving prothrombin in two places (an arg-thr andthen an arg-ile bond), which yields the active thrombin. This process isoptimized when factor Xa is complexed with activated cofactor V in theprothrombinase complex. Factor X is part of fresh frozen plasma and theprothrombinase complex. The only commercially available concentrate is“Factor X P Behring” manufactured by CSL Behring.

Factor Xa is inactivated by protein Z-dependent protease inhibitor(ZPI), a serine protease inhibitor (serpin). The affinity of thisprotein for factor Xa is increased 1000-fold by the presence of proteinZ, while it does not require protein Z for inactivation of factor XI.Defects in protein Z lead to increased factor Xa activity and apropensity for thrombosis.

Inborn deficiency of factor X is very rare (1:500,000), and may presentwith epistaxis (nosebleeds), hemarthrosis (bleeding into joints) andgastrointestinal blood loss. Apart from congenital deficiency, lowfactor X levels may occur occasionally in a number of disease states.For example, factor X deficiency may be seen in amyloidosis, wherefactor X is adsorbed to the amyloid fibrils in the vasculature. Also,deficiency of vitamin K or antagonism by warfarin (or similarmedication) leads to the production of an inactive factor X. In warfarintherapy, this is desirable to prevent thrombosis. As of late 2007, fourout of five emerging anti-coagulation therapeutics targeted this enzyme.Direct Xa inhibitors are popular anticoagulants.

Traditional models of coagulation developed in the 1960s envisaged twoseparate cascades, the extrinsic (tissue factor (TF)) pathway and theintrinsic pathway. These pathways converge to a common point, theformation of the Factor Xa/Va complex which together with calcium andbound on a phospholipids surface generate thrombin (Factor IIa) fromprothrombin (Factor II). A new model, the cell-based model ofanticoagulation appears to explain more fully the steps in coagulation.This model has three stages: 1) initiation of coagulation on TF-bearingcells, 2) amplification of the procoagulant signal by thrombin generatedon the TF-bearing cell and 3) propagation of thrombin generation on theplatelet surface. Factor Xa plays a key role in all three of thesestages.

In stage 1, Factor VII binds to the transmembrane protein TF on thesurface of cells and is converted to Factor VIIa. The result is a FactorVIIa/TF complex which catalyzes the activation of Factor X and FactorIX. Factor Xa formed on the surface of the TF-bearing cell interactswith Factor Va to form the prothrombinase complex which generates smallamounts of thrombin on the surface of TF-bearing cells. In stage 2, theamplification stage, if enough thrombin has been generated, thenactivation of platelets and platelet associated cofactors occurs. Instage 3, thrombin generation, Factor XIa activates free Factor IX on thesurface of activated platelets. The activated Factor IXa with FactorVIIIa forms the “tenase” complex. This complex activates more Factor X,which in turn forms new prothrombinase complexes with Factor Va. FactorXa is the prime component of the prothrombinase complex which convertslarge amounts of prothrombin—the “thrombin burst.” Each molecule ofFactor Xa can generate 1000 molecules of thrombin. This large burst ofthrombin is responsible for fibrin polymerization to form a thrombus.

Inhibition of the synthesis or activity of Factor X is the mechanism ofaction for many anticoagulants in use today. Warfarin, a syntheticderivative of coumarin, is the most widely used oral anticoagulant inthe U.S. In some European countries, other coumarin derivatives(phenprocoumon and acenocoumarol) are used. These agents are vitamin Kantagonists (VKA). Vitamin K is essential for the hepatic synthesis ofFactors II (prothrombin), VII, IX and X. Heparin (unfractionatedheparin) and its derivatives low molecular weight heparin (LMWH) bind toa plasma cofactor, antithrombin (AT) to inactivate several coagulationfactors IIa, Xa, XIa and XIIa.

Recently a new series of specific, direct acting inhibitors of Factor Xahas been developed. These include the drugs rivaroxaban, apixaban,betrixaban, LY517717, darexaban (YM150), edoxaban and 813893. Theseagents have several theoretical advantages over current therapy. Theymay be given orally. They have rapid onset of action. And they may bemore effective against Factor Xa in that they inhibit both free FactorXa and Factor Xa in the prothrombinase complex.

5. Protein S

Protein S is a vitamin K-dependent plasma glycoprotein synthesized inthe endothelium. In the circulation, Protein S exists in two forms: afree form and a complex form bound to complement protein C4b-bindingprotein (C4BP). In humans, Protein S is encoded by the PROS1 gene. Thebest characterized function of Protein S is its role in the anticoagulation pathway, where it functions as a cofactor to Protein C inthe inactivation of Factors Va and VIIIa. Only the free form hascofactor activity.

Protein S can bind to negatively charged phospholipids via thecarboxylated GLA domain. This property allows Protein S to function inthe removal of cells which are undergoing apoptosis. Apoptosis is a formof cell death that is used by the body to remove unwanted or damagedcells from tissues. Cells which are apoptotic (i.e., in the process ofapoptosis) no longer actively manage the distribution of phospholipidsin their outer membrane and hence begin to display negatively chargedphospholipids, such as phosphatidyl serine, on the cell surface. Inhealthy cells, an ATP (Adenosine triphosphate)-dependent enzyme removesthese from the outer leaflet of the cell membrane. These negativelycharged phospholipids are recognized by phagocytes such as macrophages.Protein S can bind to the negatively charged phospholipids and functionas a bridging molecule between the apoptotic cell and the phagocyte. Thebridging property of Protein S enhances the phagocytosis of theapoptotic cell, allowing it to be removed ‘cleanly’ without any symptomsof tissue damage such as inflammation occurring.

Mutations in the PROS1 gene can lead to Protein S deficiency which is arare blood disorder which can lead to an increased risk of thrombosis.Protein S has been shown to interact with Factor V.

6. Protein C

Protein C, also known as autoprothrombin IIA and blood coagulationfactor XIV, is a zymogenic (inactive) protein, the activated form ofwhich plays an important role in regulating blood clotting,inflammation, cell death, and maintaining the permeability of bloodvessel walls in humans and other animals. Activated protein C (APC)performs these operations primarily by proteolytically inactivatingproteins Factor V_(a) and Factor VIII_(a). APC is classified as a serineprotease as it contains a residue of serine in its active site. Inhumans, protein C is encoded by the PROC gene, which is found onchromosome 2.

The zymogenic form of protein C is a vitamin K-dependent glycoproteinthat circulates in blood plasma. Its structure is that of a two-chainpolypeptide consisting of a light chain and a heavy chain connected by adisulfide bond. The protein C zymogen is activated when it binds tothrombin, another protein heavily involved in coagulation, and proteinC's activation is greatly promoted by the presence of thrombomodulin andendothelial protein C receptors (EPCRs). Because of EPCR's role,activated protein C is found primarily near endothelial cells (i.e.,those that make up the walls of blood vessels), and it is these cellsand leukocytes (white blood cells) that APC affects. Because of thecrucial role that protein C plays as an anticoagulant, those withdeficiencies in protein C, or some kind of resistance to APC, sufferfrom a significantly increased risk of forming dangerous blood clots(thrombosis).

Research into the clinical use of activated protein C also known asdrotrecogin alfa-activated (branded Xigris) has been surrounded bycontroversy. The manufacturer Eli Lilly and Company ran an aggressivemarketing campaign to promote its use in people with severe sepsis andseptic shock including the sponsoring of the 2004 Surviving SepsisCampaign Guidelines. A 2011 Cochrane review however found that its usecannot be recommended as it does not improve survival (and increasesbleeding risk).

Human protein C is a vitamin K-dependent glycoprotein structurallysimilar to other vitamin K-dependent proteins affecting blood clotting,such as prothrombin, Factor VII, Factor IX and Factor X. Protein Csynthesis occurs in the liver and begins with a single-chain precursormolecule: a 32 amino acid N-terminus signal peptide preceding apropeptide. Protein C is formed when a dipeptide of Lys¹⁹⁸ and Arg¹⁹⁹ isremoved; this causes the transformation into a heterodimer with N-linkedcarbohydrates on each chain. The protein has one light chain (21 kDa)and one heavy chain (41 kDa) connected by a disulfide bond betweenCys¹⁸³ and Cys³¹⁹.

Inactive protein C comprises 419 amino acids in multiple domains: oneGla domain (residues 43-88); a helical aromatic segment (89-96); twoepidermal growth factor (EGF)-like domains (97-132 and 136-176); anactivation peptide (200-211); and a trypsin-like serine protease domain(212-450). The light chain contains the Gla- and EGF-like domains andthe aromatic segment. The heavy chain contains the protease domain andthe activation petide. It is in this form that 85-90% of protein Ccirculates in the plasma as a zymogen, waiting to be activated. Theremaining protein C zymogen comprises slightly modified forms of theprotein. Activation of the enzyme occurs when a thrombin moleculecleaves away the activation peptide from the N-terminus of the heavychain. The active site contains a catalytic triad typical of serineproteases (His²⁵³, Asp²⁹⁹ and Ser⁴⁰²).

The activation of protein C is strongly promoted by thrombomodulin andendothelial protein C receptor (EPCR), the latter of which is foundprimarily on endothelial cells (cells on the inside of blood vessels).The presence of thrombomodulin accelerates activation by several ordersof magnitude, and EPCR speeds up activation by a factor of 20. If eitherof these two proteins is absent in murine specimens, the mouse dies fromexcessive blood-clotting while still in an embryonic state. On theendothelium, APC performs a major role in regulating blood clotting,inflammation, and cell death (apoptosis). Because of the acceleratingeffect of thrombomodulin on the activation of protein C, the protein maybe said to be activated not by thrombin but the thrombin-thrombomodulin(or even thrombin-thrombomodulin-EPCR) complex. Once in active form, APCmay or may not remain bound to EPCR, to which it has approximately thesame affinity as the protein zymogen.

The Gla domain is particularly useful for binding to negatively-chargedphospholipids for anticoagulation and to EPCR for cytoprotection. Oneparticular exosite augments protein C's ability to inactivate FactorV_(a) efficiently. Another is necessary for interacting withthrombomodulin.

Protein C in zymogen form is present in normal adult human blood plasmaat concentrations between 65-135 IU/dL. Activated protein C is found atlevels approximately 2000 times lower than this. Mild protein Cdeficiency corresponds to plasma levels above 20 IU/dL, but below thenormal range. Moderately severe deficiencies describe bloodconcentrations between 1 and 20 IU/dL; severe deficiencies yield levelsof protein C that are below 1 IU/dL or are undetectable. Protein Clevels in a healthy term infant average 40 IU/dL. The concentration ofprotein C increases until six months, when the mean level is 60 IU/dL;the level stays low through childhood until it reaches adult levelsafter adolescence. The half-life of activated protein C is around 15minutes.

The protein C pathways are the specific chemical reactions that controlthe level of expression of APC and its activity in the body. Protein Cis pleiotropic, with two main classes of functions: anticoagulation andcytoprotection (its direct effect on cells). Which function protein Cperforms depends on whether or not APC remains bound to EPCR after it isactivated; the anticoagulative effects of APC occur when it does not. Inthis case, protein C functions as an anticoagulant by irreversiblyproteolytically inactivating Factor V_(a) and Factor VIII_(a), turningthem into Factor V_(i) and Factor VIII_(i) respectively. When stillbound to EPCR, activated protein C performs its cytoprotective effects,acting on the effector substrate PAR-1, protease-activated receptor-1.To a degree, APC's anticoagulant properties are independent of itscytoprotective ones, in that expression of one pathway is not affectedby the existence of the other.

The activity of protein C may be down-regulated by reducing the amounteither of available thrombomodulin or of EPCR. This may be done byinflammatory cytokines, such as interleukin-1β (IL-1β) and tumornecrosis factor-α (TNF-α). Activated leukocytes release theseinflammatory mediators during inflammation, inhibiting the creation ofboth thrombomodulin and EPCR, and inducing their shedding from theendothelial surface. Both of these actions down-regulate protein Cactivation. Thrombin itself may also have an effect on the levels ofEPCR. In addition, proteins released from cells can impede protein Cactivation, for example eosinophil, which may explain thrombosis inhypereosinophilic heart disease. Protein C may be up-regulated byplatelet factor 4. This cytokine is conjectured to improve activation ofprotein C by forming an electrostatic bridge from protein C's Gla domainto the glycosaminoglycan (GAG) domain of thrombomodulin, reducing theMichaelis constant (K_(M)) for their reaction. In addition, Protein C isinhibited by protein C inhibitor.

A genetic protein C deficiency, in its mild form associated with simpleheterozygosity, causes a significantly increased risk of venousthrombosis in adults. If a fetus is homozygous or compound heterozygousfor the deficiency, there may be a presentation of purpura fulminans,severe disseminated intravascular coagulation and simultaneous venousthromboembolism in the womb; this is very severe and usually fatal.Deletion of the protein C gene in mice causes fetal death around thetime of birth. Fetal mice with no protein C develop normally at first,but experience severe bleeding, coagulopathy, deposition of fibrin andnecrosis of the liver. The frequency of protein C deficiency amongasymptomatic individuals is between 1 in 200 and 1 in 500. In contrast,significant symptoms of the deficiency are detectable in 1 in 20,000individuals. No racial nor ethnic biases have been detected.

Activated protein C resistance occurs when APC is unable to perform itsfunctions. This disease has similar symptoms to protein C deficiency.The most common mutation leading to activated protein C resistance amongCaucasians is at the cleavage site in Factor V for APC. There, Arg⁵⁰⁶ isreplaced with Gln, producing Factor V Leiden. This mutation is alsocalled a R506Q. The mutation leading to the loss of this cleavage siteactually stops APC from effectively inactivating both Factor V_(a) andFactor VIII_(a). Thus, the person's blood clots too readily, and he isperpetually at an increased risk for thrombosis. Individualsheterozygous for the Factor V_(Leiden) mutation carry a risk of venousthrombosis 5-7 times higher than in the general population. Homozygoussubjects have a risk 80 times higher. This mutation is also the mostcommon hereditary risk for venous thrombosis among Caucasians.

Around 5% of APC resistance is not associated with the above mutationand Factor V_(Leiden). Other genetic mutations cause APC resistance, butnone to the extent that Factor V_(Leiden) does. These mutations includevarious other versions of Factor V, spontaneous generation ofautoantibodies targeting Factor V, and dysfunction of any of APC'scofactors. Also, some acquired conditions may reduce the efficacy of APCin performing its anticoagulative functions. Studies suggest thatbetween 20% and 60% of thrombophilic patients suffer from some form ofAPC resistance.

C. Gla Domain Peptides and Polypeptide

The present disclosure contemplates the design, production and use ofvarious Gla domain-containing peptides and polypeptides. The structuralfeatures of these molecules are as follows. First, the peptides orpolypeptides have a Gla domain containing about 30-45 consecutiveresidues comprising a Gla domain. Thus, the term “a peptide having nomore than “X” consecutive residues,” even when including the term“comprising,” cannot be understood to comprise a greater number ofconsecutive residues. Second, the peptides and polypeptides may containadditional non-Gla domain residues, such as EGF domains, Kringledomains, Fc domains, etc.

In general, the peptides and polypeptides will be 300 residues or less,again, comprising 30-45 consecutive residues of Gla domain. The overalllength may be 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225,250, 275 and up to 300 residues. Ranges of peptide length of 50-300residues, 100-300 residues, 150-300 residues 200-300, residues, 50-200residues, 100-200 residues, and 150-300 residues, and 150-200 residuesare contemplated. The number of consecutive Gla residues may be 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.

The present disclosure may utilize L-configuration amino acids,D-configuration amino acids, or a mixture thereof. While L-amino acidsrepresent the vast majority of amino acids found in proteins, D-aminoacids are found in some proteins produced by exotic sea-dwellingorganisms, such as cone snails. They are also abundant components of thepeptidoglycan cell walls of bacteria. D-serine may act as aneurotransmitter in the brain. The L and D convention for amino acidconfiguration refers not to the optical activity of the amino aciditself, but rather to the optical activity of the isomer ofglyceraldehyde from which that amino acid can theoretically besynthesized (D-glyceraldehyde is dextrorotary; L-glyceraldehyde islevorotary).

One form of an “all-D” peptide is a retro-inverso peptide. Retro-inversomodification of naturally occurring polypeptides involves the syntheticassemblage of amino acids with α-carbon stereochemistry opposite to thatof the corresponding L-amino acids, i.e., D-amino acids in reverse orderwith respect to the native peptide sequence. A retro-inverso analoguethus has reversed termini and reversed direction of peptide bonds (NH—COrather than CO—NH) while approximately maintaining the topology of theside chains as in the native peptide sequence. See U.S. Pat. No.6,261,569, incorporated herein by reference.

D. Synthesis

It will be advantageous to produce peptides and polypeptides using thesolid-phase synthetic techniques (Merrifield, 1963). Other peptidesynthesis techniques are well known to those of skill in the art(Bodanszky et al., 1976; Peptide Synthesis, 1985; Solid Phase PeptideSynthelia, 1984). Appropriate protective groups for use in suchsyntheses will be found in the above texts, as well as in ProtectiveGroups in Organic Chemistry (1973). These synthetic methods involve thesequential addition of one or more amino acid residues or suitableprotected amino acid residues to a growing peptide chain. Normally,either the amino or carboxyl group of the first amino acid residue isprotected by a suitable, selectively removable protecting group. Adifferent, selectively removable protecting group is utilized for aminoacids containing a reactive side group, such as lysine.

Using solid phase synthesis as an example, the protected or derivatizedamino acid is attached to an inert solid support through its unprotectedcarboxyl or amino group. The protecting group of the amino or carboxylgroup is then selectively removed and the next amino acid in thesequence having the complementary (amino or carboxyl) group suitablyprotected is admixed and reacted with the residue already attached tothe solid support. The protecting group of the amino or carboxyl groupis then removed from this newly added amino acid residue, and the nextamino acid (suitably protected) is then added, and so forth. After allthe desired amino acids have been linked in the proper sequence, anyremaining terminal and side group protecting groups (and solid support)are removed sequentially or concurrently, to provide the final peptide.The peptides and polypeptides of the disclosure are preferably devoid ofbenzylated or methylbenzylated amino acids. Such protecting groupmoieties may be used in the course of synthesis, but they are removedbefore the peptides and polypeptides are used. Additional reactions maybe necessary, as described elsewhere, to form intramolecular linkages torestrain conformation.

Aside from the twenty standard amino acids can can be used, there are avast number of “non-standard” amino acids. Two of these can be specifiedby the genetic code, but are rather rare in proteins. Selenocysteine isincorporated into some proteins at a UGA codon, which is normally a stopcodon. Pyrrolysine is used by some methanogenic archaea in enzymes thatthey use to produce methane. It is coded for with the codon UAG.Examples of non-standard amino acids that are not found in proteinsinclude lanthionine, 2-aminoisobutyric acid, dehydroalanine and theneurotransmitter gamma-aminobutyric acid. Non-standard amino acids oftenoccur as intermediates in the metabolic pathways for standard aminoacids—for example ornithine and citrulline occur in the urea cycle, partof amino acid catabolism. Non-standard amino acids are usually formedthrough modifications to standard amino acids. For example, homocysteineis formed through the transsulfuration pathway or by the demethylationof methionine via the intermediate metabolite S-adenosyl methionine,while hydroxyproline is made by a posttranslational modification ofproline.

E. Linkers

Linkers or cross-linking agents may be used to fuse Gla domain peptidesor polypeptides to other proteinaceous sequences (e.g., antibody Fcdomains). Bifunctional cross-linking reagents have been extensively usedfor a variety of purposes including preparation of affinity matrices,modification and stabilization of diverse structures, identification ofligand and receptor binding sites, and structural studies.Homobifunctional reagents that carry two identical functional groupsproved to be highly efficient in inducing cross-linking betweenidentical and different macromolecules or subunits of a macromolecule,and linking of polypeptide ligands to their specific binding sites.Heterobifunctional reagents contain two different functional groups. Bytaking advantage of the differential reactivities of the two differentfunctional groups, cross-linking can be controlled both selectively andsequentially. The bifunctional cross-linking reagents can be dividedaccording to the specificity of their functional groups, e.g., amino-,sulfhydryl-, guanidino-, indole-, or carboxyl-specific groups. Of these,reagents directed to free amino groups have become especially popularbecause of their commercial availability, ease of synthesis and the mildreaction conditions under which they can be applied. A majority ofheterobifunctional cross-linking reagents contains a primaryamine-reactive group and a thiol-reactive group.

In another example, heterobifunctional cross-linking reagents andmethods of using the cross-linking reagents are described in U.S. Pat.No. 5,889,155, specifically incorporated herein by reference in itsentirety. The cross-linking reagents combine a nucleophilic hydrazideresidue with an electrophilic maleimide residue, allowing coupling inone example, of aldehydes to free thiols. The cross-linking reagent canbe modified to cross-link various functional groups and is thus usefulfor cross-linking polypeptides. In instances where a particular peptidedoes not contain a residue amenable for a given cross-linking reagent inits native sequence, conservative genetic or synthetic amino acidchanges in the primary sequence can be utilized.

F. Additional Peptide/Polypeptide Sequences

One factor drug development is to achieve adequate circulatinghalf-lives, which impact dosing, drug administration and efficacy, andthis has particular important to biotherapeutics. Small proteins below60 kD are cleared rapidly by the kidney and therefore do not reach theirtarget. This means that high doses are needed to reach efficacy. Themodifications currently used to increase the half-life of proteins incirculation include: PEGylation; conjugation or genetic fusion withproteins, e.g., transferrin (WO06096515A2), albumin, growth hormone(U.S. Patent Publication 2003104578AA); conjugation with cellulose (Levyand Shoseyov, 2002); conjugation or fusion with Fc fragments;glycosylation and mutagenesis approaches (Carter, 2006).

In the case of PEGylation, polyethylene glycol (PEG) is conjugated tothe protein, which can be for example a plasma protein, antibody orantibody fragment. The first studies regarding the effect of PEGylationof antibodies were performed in the 1980s. The conjugation can be doneeither enzymatically or chemically and is well established in the art(Chapman, 2002; Veronese and Pasut, 2005). With PEGylation the totalsize can be increased, which reduces the chance of renal filtration.PEGylation further protects from proteolytic degradation and slows theclearance from the blood. Further, it has been reported that PEGylationcan reduce immunogenicity and increase solubility. The improvedpharmacokinetics by the addition of PEG is due to several differentmechanisms: increase in size of the molecule, protection fromproteolysis, reduced antigenicity, and the masking of specific sequencesfrom cellular receptors. In the case of antibody fragments (Fab), a20-fold increase in plasma half-life has been achieved by PEGylation(Chapman, 2002).

To date there are several approved PEGylated drugs, e.g., PEG-interferonalpha2b (PEG-INTRON) marketed in 2000 and alpha2a (Pegasys) marketed in2002. A PEGylated antibody fragment against TNF alpha, called Cimzia orCertolizumab Pegol, was filed for FDA approval for the treatment ofCrohn's disease in 2007 and has been approved on Apr. 22, 2008. Alimitation of PEGylation is the difficulty in synthesizing longmonodisperse species, especially when PEG chains over 1000 kD areneeded. For many applications, polydisperse PEG with a chain length over10000 kD is used, resulting in a population of conjugates havingdifferent length PEG chains, which need extensive analytics to ensureequivalent batches between productions. The different length of the PEGchains may result in different biological activities and thereforedifferent pharmacokinetics. Another limitation of PEGylation is adecrease in affinity or activity as it has been observed withalpha-interferon Pegasys, which has only 7% of the antiviral activity ofthe native protein, but has improved pharmacokinetics due to theenhanced plasma half-life.

Another approach is to conjugate the drug with a long lived protein,e.g., albumin, which is 67 kD and has plasma half-life of 19 days inhuman. Albumin is the most abundant protein in plasma and is involved inplasma pH regulation, but also serves as a carrier of substances inplasma. In the case of CD4, increased plasma half-life has been achievedafter fusing it to human serum albumin (Yeh et al., 1992). Otherexamples for fusion proteins are insulin, human growth hormone,transferrin and cytokines (Duttaroy et al., 2005; Melder et al., 2005;Osborn et al., 2002a; Osborn et al., 2002b; Sung et al., 2003) and see(U.S. Patent Publication 2003104578A1, WO06096515A2, and WO07047504A2,herein incorporated in entirety by reference).

The effect of glycosylation on plasma half-life and protein activity hasalso been extensively studied. In the case of tissue plasminogenactivator (tPA), the addition of new glycosylation sites decreased theplasma clearance, and improved the potency (Keyt et al., 1994).Glycoengineering has been successfully applied for a number ofrecombinant proteins and immunoglobulins (Elliott et al., 2003; Raju andScallon, 2007; Sinclair and Elliott, 2005; Umana et al., 1999). Further,glycosylation influences the stability of immunoglobulins (Mimura etal., 2000; Raju and Scallon, 2006).

Another molecule used for fusion proteins is the Fc fragment of an IgG(Ashkenazi and Chamow, 1997). The Fc fusion approach has been utilized,for example in the Trap Technology developed by Regeneron (e.g., IL1trap and VEGF trap). The use of albumin to extend the half-life ofpeptides has been described in U.S. Patent Publication 2004001827A1.Positive effects of albumin have also been reported for Fab fragmentsand scFv-HSA fusion protein. It has been demonstrated that the prolongedserum half-life of albumin is due to a recycling process mediated by theFcRn (Anderson et al., 2006; Chaudhury et al., 2003).

Another strategy is to use directed mutagenesis techniques targeting theinteraction of immunoglobulins to their receptor to improve bindingproperties, i.e., affinity maturation in the Fc region. With anincreased affinity to FcRn a prolonged half-life can be achieved in vivo(Ghetie et al., 1997; Hinton et al., 2006; Jain et al., 2007; Petkova etal., 2006a; Vaccaro et al., 2005). However, affinity maturationstrategies require several rounds of mutagenesis and testing. This takestime, is costly and is limited by the number of amino acids that whenmutated result in prolonged half-lives. Therefore, simple alternativeapproaches are needed to improve the in vivo half-life ofbiotherapeutics. Therapeutics with extended half-lives in vivo areespecially important for the treatment of chronic diseases, autoimmunedisorders, inflammatory, metabolic, infectious, and eye diseases, andcancer, especially when therapy is required over a long time period.Accordingly, a need still exists for the development of therapeuticagents (e.g., antibodies and Fc fusion proteins) with enhancedpersistence and half-lives in circulation, in order to reduce the dosageand/or the frequency of injections of a variety of therapeutic agents.

G. Labels

The peptides and polypeptides of the present disclosure may beconjugated to labels for diagnostic purposes. A label in accordance withthe present disclosure is defined as any moiety which may be detectedusing an assay. Non-limiting examples of reporter molecules includeenzymes, radiolabels, haptens, fluorescent labels, phosphorescentmolecules, chemiluminescent molecules, chromophores, photoaffinitymolecules, colored particles or ligands, such as biotin.

Label conjugates are generally preferred for use as diagnostic agents.Diagnostic agents generally fall within two classes, those for use in invitro diagnostics, and those for use in vivo diagnostic protocols,generally known as “directed imaging.” Many appropriate imaging agentsare known in the art, as are methods for their attachment to peptidesand polypeptides (see, for e.g., U.S. Pat. Nos. 5,021,236, 4,938,948,and 4,472,509). The imaging moieties used can be paramagnetic ions,radioactive isotopes, fluorochromes, NMR-detectable substances, andX-ray imaging agents.

In the case of paramagnetic ions, one might mention by way of exampleions such as chromium (III), manganese (II), iron (III), iron (II),cobalt (II), nickel (II), copper (II), neodymium (III), samarium (III),ytterbium (III), gadolinium (III), vanadium (II), terbium (III),dysprosium (III), holmium (III) and/or erbium (III), with gadoliniumbeing particularly preferred. Ions useful in other contexts, such asX-ray imaging, include but are not limited to lanthanum (III), gold(III), lead (II), and especially bismuth (III).

In the case of radioactive isotopes for therapeutic and/or diagnosticapplication, one might mention astatine²¹¹, ¹⁴carbon, ⁵¹chromium,³⁶chlorine, ⁵⁷cobalt, ⁵⁸cobalt, copper⁶⁷, ¹⁵²Eu, gallium⁶⁷, ³hydrogen,iodine¹²³, iodine¹²⁵, iodine¹³¹, indium¹¹¹, ⁵⁹iron, ³²phosphorus,rhenium¹⁸⁶, rhenium¹⁸⁸, ⁷⁵selenium, ³⁵sulphur, technicium^(99m) and/oryttrium⁹⁰, ¹²⁵I is often being preferred for use in certain embodiments,and technicium^(99m) and/or indium¹¹¹ are also often preferred due totheir low energy and suitability for long range detection. Radioactivelylabeled peptides and polypeptides may be produced according towell-known methods in the art. For instance, peptides and polypeptidescan be iodinated by contact with sodium and/or potassium iodide and achemical oxidizing agent such as sodium hypochlorite, or an enzymaticoxidizing agent, such as lactoperoxidase. Peptides may be labeled withtechnetium^(99m) by ligand exchange process, for example, by reducingpertechnate with stannous solution, chelating the reduced technetiumonto a Sephadex column and applying the peptide to this column.Alternatively, direct labeling techniques may be used, e.g., byincubating pertechnate, a reducing agent such as SNCl₂, a buffersolution such as sodium-potassium phthalate solution, and the peptide.Intermediary functional groups which are often used to bindradioisotopes which exist as metallic ions to peptide arediethylenetriaminepentaacetic acid (DTPA) or ethylene diaminetetraceticacid (EDTA).

Among the fluorescent labels contemplated for use as conjugates includeAlexa 350, Alexa 430, AMCA, BODIPY 630/650, BODIPY 650/665, BODIPY-FL,BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, Cascade Blue, Cy3, Cy5,6-FAM,Fluorescein Isothiocyanate, HEX, 6-JOE, Oregon Green 488, Oregon Green500, Oregon Green 514, Pacific Blue, REG, Rhodamine Green, RhodamineRed, Renographin, ROX, TAMRA, TET, Tetramethylrhodamine, and/or TexasRed.

Another type of conjugate contemplated is that intended primarily foruse in vitro, where the peptide is linked to a secondary binding ligandand/or to an enzyme (an enzyme tag) that will generate a colored productupon contact with a chromogenic substrate. Examples of suitable enzymesinclude urease, alkaline phosphatase, (horseradish) hydrogen peroxidaseor glucose oxidase. Preferred secondary binding ligands are biotin andavidin and streptavidin compounds. The use of such labels is well knownto those of skill in the art and is described, for example, in U.S. Pat.Nos. 3,817,837, 3,850,752, 3,939,350, 3,996,345, 4,277,437, 4,275,149and 4,366,241.

Other methods are known in the art for the attachment or conjugation ofa peptide to its conjugate moiety. Some attachment methods involve theuse of a metal chelate complex employing, for example, an organicchelating agent such a diethylenetriaminepentaacetic acid anhydride(DTPA); ethylenetriaminetetraacetic acid; N-chloro-p-toluenesulfonamide;and/or tetrachloro-3α-6α-diphenylglycouril-3 attached to the antibody(U.S. Pat. Nos. 4,472,509 and 4,938,948). Peptides or polypeptides mayalso be reacted with an enzyme in the presence of a coupling agent suchas glutaraldehyde or periodate. Conjugates with fluorescein markers areprepared in the presence of these coupling agents or by reaction with anisothiocyanate.

IV. THERAPIES

A. Pharmaceutical Formulations and Routes of Administration

Where clinical applications are contemplated, it will be necessary toprepare pharmaceutical compositions in a form appropriate for theintended application. Generally, this will entail preparing compositionsthat are essentially free of pyrogens, as well as other impurities thatcould be harmful to humans or animals.

One will generally desire to employ appropriate salts and buffers torender delivery vectors stable and allow for uptake by target cells.Buffers also will be employed when recombinant cells are introduced intoa patient. Aqueous compositions of the present disclosure comprise aneffective amount of the vector to cells, dissolved or dispersed in apharmaceutically acceptable carrier or aqueous medium. Such compositionsalso are referred to as inocula. The phrase “pharmaceutically orpharmacologically acceptable” refer to molecular entities andcompositions that do not produce adverse, allergic, or other untowardreactions when administered to an animal or a human. As used herein,“pharmaceutically acceptable carrier” includes any and all solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents and the like. The use of suchmedia and agents for pharmaceutically active substances is well known inthe art. Except insofar as any conventional media or agent isincompatible with the vectors or cells of the present disclosure, itsuse in therapeutic compositions is contemplated. Supplementary activeingredients also can be incorporated into the compositions.

The active compositions of the present disclosure may include classicpharmaceutical preparations. Administration of these compositionsaccording to the present disclosure will be via any common route so longas the target tissue is available via that route. Such routes includeoral, nasal, buccal, rectal, vaginal or topical route. Alternatively,administration may be by orthotopic, intradermal, subcutaneous,intramuscular, intratumoral, intraperitoneal, or intravenous injection.Such compositions would normally be administered as pharmaceuticallyacceptable compositions, described supra.

The active compounds may also be administered parenterally orintraperitoneally. Solutions of the active compounds as free base orpharmacologically acceptable salts can be prepared in water suitablymixed with a surfactant, such as hydroxypropylcellulose. Dispersions canalso be prepared in glycerol, liquid polyethylene glycols, and mixturesthereof and in oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases the form must be sterile and must be fluid tothe extent that easy syringability exists. It must be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms, such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), suitable mixtures thereof,and vegetable oils. The proper fluidity can be maintained, for example,by the use of a coating, such as lecithin, by the maintenance of therequired particle size in the case of dispersion and by the use ofsurfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial and antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, andthe like. In many cases, it will be preferable to include isotonicagents, for example, sugars or sodium chloride. Prolonged absorption ofthe injectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminummonostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousother ingredients as enumerated above, as required, followed by filteredsterilization. Generally, dispersions are prepared by incorporating thevarious sterilized active ingredients into a sterile vehicle whichcontains the basic dispersion medium and the required other ingredientsfrom those enumerated above. In the case of sterile powders for thepreparation of sterile injectable solutions, the preferred methods ofpreparation are vacuum-drying and freeze-drying techniques which yield apowder of the active ingredient plus any additional desired ingredientfrom a previously sterile-filtered solution thereof.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents and the like. The use ofsuch media and agents for pharmaceutical active substances is well knownin the art. Except insofar as any conventional media or agent isincompatible with the active ingredient, its use in the therapeuticcompositions is contemplated. Supplementary active ingredients can alsobe incorporated into the compositions.

For oral administration the peptides and polypeptides of the presentdisclosure may be incorporated with excipients and used in the form ofnon-ingestible mouthwashes and dentifrices. A mouthwash may be preparedincorporating the active ingredient in the required amount in anappropriate solvent, such as a sodium borate solution (Dobell'sSolution). Alternatively, the active ingredient may be incorporated intoan antiseptic wash containing sodium borate, glycerin and potassiumbicarbonate. The active ingredient may also be dispersed in dentifrices,including: gels, pastes, powders and slurries. The active ingredient maybe added in a therapeutically effective amount to a paste dentifricethat may include water, binders, abrasives, flavoring agents, foamingagents, and humectants.

The compositions of the present disclosure may be formulated in aneutral or salt form. Pharmaceutically-acceptable salts include the acidaddition salts (formed with the free amino groups of the protein) andwhich are formed with inorganic acids such as, for example, hydrochloricor phosphoric acids, or such organic acids as acetic, oxalic, tartaric,mandelic, and the like. Salts formed with the free carboxyl groups canalso be derived from inorganic bases such as, for example, sodium,potassium, ammonium, calcium, or ferric hydroxides, and such organicbases as isopropylamine, trimethylamine, histidine, procaine and thelike.

Upon formulation, solutions will be administered in a manner compatiblewith the dosage formulation and in such amount as is therapeuticallyeffective. The formulations are easily administered in a variety ofdosage forms such as injectable solutions, drug release capsules and thelike. For parenteral administration in an aqueous solution, for example,the solution should be suitably buffered if necessary and the liquiddiluent first rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous and intraperitoneal administration. In thisconnection, sterile aqueous media which can be employed will be known tothose of skill in the art in light of the present disclosure. Forexample, one dosage could be dissolved in 1 ml of isotonic NaCl solutionand either added to 1000 ml of hypodermoclysis fluid or injected at theproposed site of infusion, (see for example, “Remington's PharmaceuticalSciences,” 15th Edition, pages 1035-1038 and 1570-1580). Some variationin dosage will necessarily occur depending on the condition of thesubject being treated. The person responsible for administration will,in any event, determine the appropriate dose for the individual subject.Moreover, for human administration, preparations should meet sterility,pyrogenicity, general safety and purity standards as required by FDAOffice of Biologics standards.

B. Disease States and Conditions

1. Cancer

Background.

Cancer results from the outgrowth of a clonal population of cells fromtissue. The development of cancer, referred to as carcinogenesis, can bemodeled and characterized in a number of ways. An association betweenthe development of cancer and inflammation has long-been appreciated.The inflammatory response is involved in the host defense againstmicrobial infection, and also drives tissue repair and regeneration.Considerable evidence points to a connection between inflammation and arisk of developing cancer, i.e., chronic inflammation can lead todysplasia. There are hundreds of different forms of human cancers, andwith an increasing understanding of the underlying genetics and biologyof cancer, these forms are being further subdivided and reclassified.

Determining what causes cancer is complex. Many things are known toincrease the risk of cancer, including tobacco use, certain infections,radiation, lack of physical activity, obesity, and environmentalpollutants. These can directly damage genes or combine with existinggenetic faults within cells to cause the disease. Approximately five toten percent of cancers are entirely hereditary.

Cancer can be detected in a number of ways, including the presence ofcertain signs and symptoms, screening tests, or medical imaging. Once apossible cancer is detected it is diagnosed by microscopic examinationof a tissue sample. Cancer is usually treated with chemotherapy,radiation therapy and surgery. The chances of surviving the disease varygreatly by the type and location of the cancer and the extent of diseaseat the start of treatment. While cancer can affect people of all ages,and a few types of cancer are more common in children, the risk ofdeveloping cancer generally increases with age. In 2007, cancer causedabout 13% of all human deaths worldwide (7.9 million). Rates are risingas more people live to an old age and as mass lifestyle changes occur inthe developing world.

Treatments fall in to five general categories: surgery, chemotherapy,radiation, alternative medicine and palliative care. Surgery is theprimary method of treatment of most isolated solid cancers and may playa role in palliation and prolongation of survival. It is typically animportant part of making the definitive diagnosis and staging the tumoras biopsies are usually required. In localized cancer surgery typicallyattempts to remove the entire mass along with, in certain cases, thelymph nodes in the area. For some types of cancer this is all that isneeded to eliminate the cancer.

Chemotherapy in addition to surgery has proven useful in a number ofdifferent cancer types including: breast cancer, colorectal cancer,pancreatic cancer, osteogenic sarcoma, testicular cancer, ovariancancer, and certain lung cancers. The effectiveness of chemotherapy isoften limited by toxicity to other tissues in the body.

Radiation therapy involves the use of ionizing radiation in an attemptto either cure or improve the symptoms of cancer. It is used in abouthalf of all cases and the radiation can be from either internal sourcesin the form of brachytherapy or external sources. Radiation is typicallyused in addition to surgery and or chemotherapy but for certain types ofcancer such as early head and neck cancer may be used alone. For painfulbone metastasis it has been found to be effective in about 70% ofpeople.

Alternative and complementary treatments include a diverse group ofhealth care systems, practices, and products that are not part ofconventional medicine “Complementary medicine” refers to methods andsubstances used along with conventional medicine, while “alternativemedicine” refers to compounds used instead of conventional medicine.Most complementary and alternative medicines for cancer have not beenrigorously studied or tested. Some alternative treatments have beeninvestigated and shown to be ineffective but still continue to bemarketed and promoted.

Finally, palliative care refers to treatment which attempts to make thepatient feel better and may or may not be combined with an attempt toattack the cancer. Palliative care includes action to reduce thephysical, emotional, spiritual, and psycho-social distress experiencedby people with cancer. Unlike treatment that is aimed at directlykilling cancer cells, the primary goal of palliative care is to improvethe patient's quality of life.

Gla Domain Activity.

In the context of the present disclosure, it is contemplated that theengineered Gla proteins may be used as anti-cancer agents in severaldifferent fashions. First, cancer patients often suffer from ahypercoagulable state which can lead to embolism and death. In thisembodiment, the Gla domain proteins (optionally linked to an Fc region)are administered into the vasculature of the subject in amountssufficient to reduce emboli formation and pathological coagulation. SuchGla domain proteins disclosed herein are useful in reducing, blockingand/or inhibiting pathologic coagulation in such patients.

Also, it is known that PtdS plays a role in suppressing the immuneresponse to cancer. Thus, in another embodiment, the inventors envisionthe use of Gla domain proteins to bind and block/mask PtdS on cancercells. This serves to prevent tolerogenic signaling through PtdS thatinduces anti-inflammatory cytokines and suppresses pro-inflammatorycytokines, thereby facilitating immune escape of the cancer cells. Thismay prove a particularly useful approach when combined with other“standard” cancer therapeutics, as discussed in detail below.

2. Autoimmune/Inflammatory Disease

Background.

The present disclosure contemplates the treatment of a variety ofautoimmune and/or inflammatory disease states such asspondyloarthropathy, ankylosing spondylitis, psoriatic arthritis,reactive arthritis, enteropathic arthritis, ulcerative colitis, Crohn'sdisease, irritable bowel disease, inflammatory bowel disease, rheumatoidarthritis, juvenile rheumatoid arthritis, familial Mediterranean fever,amyotrophic lateral sclerosis, Sjogren's syndrome, early arthritis,viral arthritis, multiple sclerosis, systemic lupus erythematosus,psoriasis, vasculitis, Wegener's granulomatosis, Addison's disease,alopecia, antiphospholipid syndrome, Behcet's disease, celiac disease,chronic fatigue syndrome, ulcerative colitis, type I diabetes,fibromyalgia, autoimmune gastritis, Goodpasture syndrome, Graves'disease, idiopathic thrombocytopenic purpura (ITP), myasthenia gravis,pemphigus vulgaris, primary biliary cirrhosis, rheumatic fever,sarcoidosis, scleroderma, vitiligo, vasculitis, small vessel vasculitis,hepatitis, primary biliary cirrhosis, sarcoidosis, scleroderma, graftversus host disease (acute and chronic), aplastic anemia, or cyclicneutropenia. The diagnosis and treatment of these diseases are welldocumented in the literature.

Gla Domain Activity.

It is known that certain immune disorders are characterized by deficientphagocytosis of apoptotic cells, leading to secondary necrosis,pro-inflammatory signaling and autoantibody production. Further, plasmamembrane-derived vesicles (PMVs) containing PtdS on their surface areshed from apoptotic cells and are thought to bind to immune cells, suchas macrophages (DeRose et al., 2011). Gla domain proteins can be used tomask the PtdS found on the surface of PMVs, thereby reducing the “decoy”effect of the PMVs and permitting phagocytosis of apoptotic cells. Thisshould break the cycle of continued inflammatory signaling and reduceimmune hyperactivation.

3. Hypercoagulation Disorders

Background.

Hypercoagulation disorders give rise to an increased tendency forclotting of the blood, which put a patient at risk for obstruction ofveins and arteries. In normal hemostasis, or the stoppage of bleeding,clots form at the site of the blood vessel's injury. Inhypercoagulation, clots develop in circulating blood. When this occursthroughout the body's blood vessels, a condition known as thrombosis canarise. Thrombosis can lead to infarction, or death of tissue.Hypercoagulation disorders include hyperhomocystinemia, antithrombin IIIdeficiency, factor V leyden, and protein C or protein S deficiency.

The diagnosis of hypercoagulation disorders is completed with acombination of physical examination, medical history, and blood tests.When found, coumadin and heparin anticoagulants may be administered toreduce the clotting effects and maintain fluidity in the blood. Theprognosis for patients with hypercoagulation disorders varies dependingon the severity of the clotting and thrombosis, but if undetected anduntreated, thrombosis could lead to pulmonary embolism and death.

Sickle cell disease (SCD), though not a clotting disorder, has some ofthe same manifestations. Interestingly, an increased level of surfaceexpressed PtdS on a subpopulation of erythrocytes is a universal featureof this disease (Wood et al., Blood, Vol 88, No 5 (September 1), 1996).SCD is an autosomal recessive genetic blood disorder with overdominance,characterized by red blood cells that assume an abnormal, rigid, sickleshape. Sickling decreases the cells' flexibility and results in a riskof various complications. The sickling occurs because of a mutation inthe hemoglobin gene. Life expectancy is shortened. Sickle-cell diseaseoccurs more commonly in people (or their descendants) from parts oftropical and sub-tropical sub-Saharan regions where malaria is or wascommon. In areas where malaria is common, there is a fitness benefit incarrying only a single sickle-cell gene (sickle cell trait). Those withonly one of the two alleles of the sickle-cell disease, while nottotally resistant, are more tolerant to the infection and thus show lesssevere symptoms when infected.

Sickle-cell anaemia is the name of a specific form of sickle-celldisease in which there is homozygosity for the mutation that causes HbS.Sickle-cell anaemia is also referred to as “HbSS,” “SS disease,”“hemoglobin S” or permutations thereof. In heterozygous people, who haveonly one sickle gene and one normal adult haemoglobin gene, it isreferred to as “HbAS” or “sickle cell trait.” Other, rarer forms ofsickle-cell disease include sickle-hemoglobin C disease (HbSC), sicklebeta-plus-thalassemia (HbS/β⁺) and sicklebeta-zero-thalassaemia)(HbS/β⁰). These other forms of sickle-celldisease are compound heterozygous states in which the person has onlyone copy of the mutation that causes HbS and one copy of anotherabnormal hemoglobin allele.

In particular aspects, SCD can relate to blood flow. The vaso-occlusivecrisis is caused by sickle-shaped red blood cells that obstructcapillaries and restrict blood flow to an organ, resulting in ischemia,pain, necrosis and often organ damage. The frequency, severity, andduration of these crises vary considerably. Painful crises are treatedwith hydration, analgesics, and blood transfusion; pain managementrequires opioid administration at regular intervals until the crisis hassettled. For milder crises, a subgroup of patients manage on NSAIDs(such as diclofenac or naproxen). For more severe crises, most patientsrequire inpatient management for intravenous opioids; patient-controlledanalgesia (PCA) devices are commonly used in this setting.Vaso-occlusive crisis involving organs such as the penis or lungs areconsidered an emergency and treated with red-blood cell transfusions.Diphenhydramine is sometimes effective for the itching associated withthe opioid use. Incentive spirometry, a technique to encourage deepbreathing to minimize the development of atelectasis, is recommended.

Gla Domain Activity.

The present invention contemplates the use of Gla domains to control ormodulate the pro-coagulant actions of PtdS in hypercoagulation. PtdSsurface expression is a potent stimulator of the coagulation cascade.Masking exposed PtdS via the use of Gla domain polypeptides couldinhibit or dampen this cascade. This treatment could be particularlybeneficial for SCA individuals where it is believed that only a smallpopulation of RBCs may be responsible for the hypercoagulabilityassociated with the disease

4. Viral Infection

Background.

A virus is a small infectious agent that can replicate only inside theliving cells of an organism. Viruses can infect all types of organisms,from animals and plants to bacteria and archaea. About 5,000 viruseshave been described in detail, although there are millions of differenttypes. Viruses are found in almost every ecosystem on Earth and are themost abundant type of biological entity.

Virus particles (known as virions) consist of two or three parts: i) thegenetic material made from either DNA or RNA, long molecules that carrygenetic information; ii) a protein coat that protects these genes; andin some cases iii) an envelope of lipids that surrounds the protein coatwhen they are outside a cell. The shapes of viruses range from simplehelical and icosahedral forms to more complex structures. The averagevirus is about one one-hundredth the size of the average bacterium. Mostviruses are too small to be seen directly with an optical microscope.

Viruses spread in many ways; viruses in plants are often transmittedfrom plant to plant by insects that feed on plant sap, such as aphids;viruses in animals can be carried by blood-sucking insects. Thesedisease-bearing organisms are known as vectors. Influenza viruses arespread by coughing and sneezing. Norovirus and rotavirus, common causesof viral gastroenteritis, are transmitted by the fecal-oral route andare passed from person to person by contact, entering the body in foodor water. HIV is one of several viruses transmitted through sexualcontact and by exposure to infected blood. The range of host cells thata virus can infect is called its “host range.” This can be narrow or, aswhen a virus is capable of infecting many species, broad.

Viral infections in animals provoke an immune response that usuallyeliminates the infecting virus Immune responses can also be produced byvaccines, which confer an artificially acquired immunity to the specificviral infection. However, some viruses including those that cause AIDSand viral hepatitis evade these immune responses and result in chronicinfections. Antibiotics have no effect on viruses, but several antiviraldrugs have been developed.

A variety of diseases are fostered by virus infections, includinginfluenza, human immunodeficiency virus, dengue virus, West Nile virus,smallpox virus, respiratory syncytial virus, Korean hemorrhagic fevervirus, chickenpox, varicella zoster virus, herpes simplex virus 1 or 2,Epstein-Barr virus, Marburg virus, hantavirus, yellow fever virus,hepatitis A, B, C or E, Ebola virus, human papilloma virus, rhinovirus,Coxsackie virus, polio virus, measles virus, rubella virus, rabiesvirus, Newcastle disease virus, rotavirus, HTLV-1 and -2.

Gla Domain Activity.

PtdS is segregated to the inner leaflet of the plasma membrane ofresting mammalian cells. Loss of phosphatidylserine asymmetry occursduring apoptosis, cell injury and cell activation such as observedduring viral infection. This results from inhibition of the translocasesor activation of phosphatidylserine exporters, or lipid-scramblingenzymes, such as scramblases (Soares et al., 2008). Viruses activatehost cells to replicate efficiently, and virus-induced cell activationmay lead to rises in intracellular Ca²⁺ that, in turn, causephosphatidylserine externalization by activating phosphatidylserineexporters and by inhibiting phosphatidylserine import by translocases.Moreover, phosphatidylserine translocation to the external host cellsurface could be an early event associated with virus-induced apoptosis(Soares et al., 2008).

PtdS on the surface of infected cells, budding virions or microvesiclesderived from pre-apoptotic cells leads to immune evasion. Using the Gladomain polypeptides of the present disclosure to mask the PtdS on thesemembranes, normal immune function can more effectively address the viralchallenge, thus reducing viral load, limiting infection, and destroyingvirions and infected cells.

5. Sepsis

Background.

In sepsis, inflammation and thrombosis are both cause and result ofinteractions between circulating (for example, leukocytes andplatelets), endothelial and smooth muscle cells. Microparticles arepro-inflammatory and procoagulant fragments originating from plasmamembrane generated after cellular activation and released in bodyfluids. In the vessel, they constitute a pool of bioactive effectorspulled from diverse cellular origins and may act as intercellularmessengers. Microparticles expose phosphatidylserine, a procoagulantphospholipid made accessible after membrane remodeling, and tissuefactor (TF), the initiator of blood coagulation at the endothelial andleukocyte surface. They constitute a secretion pathway for IL-1β andupregulate the pro-inflammatory response of target cells. Microparticlescirculate at low levels in healthy individuals, but undergo phenotypicand quantitative changes that could play a pathophysiological role ininflammatory diseases. Microparticles (MPs) may participate in thepathogenesis of sepsis through multiple ways. They are able to regulatevascular tone and are potent vascular pro-inflammatory and pro-coagulantmediators. Microparticles' abilities are of increasing interest indeciphering the mechanisms underlying the multiple organ dysfunction ofseptic shock.

The plasma membrane of endothelial cells and monocytes is reorganizedwith externalisation of phosphatidylserine and encrypted tissue factorTF expression, allowing factor VII (FVIIa) activation and thrombin(FIIa) generation at the cell surface. Blebbing occurs, with release ofmicroparticles bearing TF, resulting in an increased surface forprocoagulant reactions. Platelet adhesion and aggregation also occurwith the release of MPs; platelets and MPs bear GPIbα, a cofactor forfactor XI activation by thrombin, leading to the propagation phase withhigh levels of thrombin generation and fibrin formation. EndothelialTF-bearing MPs allow transfer of TF to PMNs, increasing TF disseminationand thrombotic microangiopathy or disseminated intravascularcoagulopathy. TF initiation of blood coagulation is quicklydown-regulated by tissue factor pathway inhibitor (TFPI) on endothelialand monocytic cell surfaces, as on MPs. Endothelial protein C receptor(EPCR)-bound protein C is activated by the thrombin-thrombomodulincomplex and activated protein C (APC) inhibits factor Va and factorVIIIa, limiting the propagation phase of thrombin generation. EPCR-boundAPC also regulates NF-KB, with cytoprotective effects on endothelialcells and monocytes. APC induces blebbing, with emission of EPCR-bearingMPs able to activate protein C, resulting in the dissemination ofanticoagulant and antiapoptotic activities.

Gla Domain Activity.

The present invention contemplates the use of Gla domains to control ormodulate the pro-coagulant actions of microparticles containing PtdS andTF on their surface. They can also down-regulate the relatedpro-inflammatory signaling that occurs in sepsis, for example, throughIL-1β, as well as modulating the effects of PtdS-bearing microparticleson vascular tone.

C. Treatment Methods

Peptides and polypeptides can be administered to mammalian subjects(e.g., human patients) alone or in conjunction with other drugs thattreat the diseases set forth above. The dosage required depends on thechoice of the route of administration; the nature of the formulation,including additional agents attached to the polypeptide; the nature ofthe patient's illness; the subject's size, weight, surface area, age,and sex; further combination therapies; and the judgment of theattending physician. Suitable dosages are in the range of 0.0001-100mg/kg. Wide variations in the needed dosage are to be expected in viewof the variety of compounds available and the differing efficiencies ofvarious routes of administration. For example, oral administration wouldbe expected to require higher dosages than administration by intravenousinjection. Variations in these dosage levels can be adjusted usingstandard empirical routines for optimization as is well understood inthe art. Administrations can be single or multiple (e.g., 2-, 3-, 4-,6-, 8-, 10-, 20-, 50-, 100-, 150-, or more times). Encapsulation of thepolypeptide in a suitable delivery vehicle (e.g., polymericmicroparticles or implantable devices) may increase the efficiency ofdelivery, particularly for oral delivery.

D. Combination Therapies

It is common in many fields of medicine to treat a disease with multipletherapeutic modalities, often called “combination therapies.” To treatdisorders using the methods and compositions of the present disclosure,one would generally contact a target cell or subject with a Gla domainpolypeptide and at least one other therapy. These therapies would beprovided in a combined amount effective to achieve a reduction in one ormore disease parameter. This process may involve contacting thecells/subjects with the both agents/therapies at the same time, e.g.,using a single composition or pharmacological formulation that includesboth agents, or by contacting the cell/subject with two distinctcompositions or formulations, at the same time, wherein one compositionincludes the Gla domain polypeptide and the other includes the otheragent.

Alternatively, the Gla domain polypeptide may precede or follow theother treatment by intervals ranging from minutes to weeks. One wouldgenerally ensure that a significant period of time did not expirebetween the time of each delivery, such that the therapies would stillbe able to exert an advantageously combined effect on the cell/subject.In such instances, it is contemplated that one would contact the cellwith both modalities within about 12-24 hours of each other, withinabout 6-12 hours of each other, or with a delay time of only about 12hours. In some situations, it may be desirable to extend the time periodfor treatment significantly; however, where several days (2, 3, 4, 5, 6or 7) to several weeks (1, 2, 3, 4, 5, 6, 7 or 8) lapse between therespective administrations.

It also is conceivable that more than one administration of either theGla domain protein or the other therapy/agent will be desired. Variouscombinations may be employed, where the Gla domain protein is “A,” andthe other therapy is “B,” as exemplified below:

A/B/A B/A/B B/B/A A/A/B B/A/A A/B/B B/B/B/A B/B/A/B A/A/B/B A/B/A/BA/B/B/A B/B/A/A B/A/B/A B/A/A/B B/B/B/A A/A/A/B B/A/A/A A/B/A/A A/A/B/AA/B/B/B B/A/B/B B/B/A/BOther combinations are contemplated.

Agents or factors suitable for use in a combined therapy against acancer include radionuclides, chemotherapeutic agents or toxins.Specific chemotherapeutics include temozolomide, epothilones, melphalan,carmustine, busulfan, lomustine, cyclophosphamide, dacarbazine,polifeprosan, ifosfamide, chlorambucil, mechlorethamine, busulfan,cyclophosphamide, carboplatin, cisplatin, thiotepa, capecitabine,streptozocin, bicalutamide, flutamide, nilutamide, leuprolide acetate,doxorubicin hydrochloride, bleomycin sulfate, daunorubicinhydrochloride, dactinomycin, liposomal daunorubicin citrate, liposomaldoxorubicin hydrochloride, epirubicin hydrochloride, idarubicinhydrochloride, mitomycin, doxorubicin, valrubicin, anastrozole,toremifene citrate, cytarabine, fluorouracil, fludarabine, floxuridine,interferon α-2b, plicamycin, mercaptopurine, methotrexate, interferonα-2a, medroxyprogersterone acetate, estramustine phosphate sodium,estradiol, leuprolide acetate, megestrol acetate, octreotide acetate,deithylstilbestrol diphosphate, testolactone, goserelin acetate,etoposide phosphate, vincristine sulfate, etoposide, vinblastine,etoposide, vincristine sulfate, teniposide, trastuzumab, gemtuzumabozogamicin, rituximab, exemestane, irinotecan hydrocholride,asparaginase, gemcitabine hydrochloride, altretamine, topotecanhydrochloride, hydroxyurea, cladribine, mitotane, procarbazinehydrochloride, vinorelbine tartrate, pentrostatin sodium, mitoxantrone,pegaspargase, denileukin diftitix, altretinoin, porfimer, bexarotene,paclitaxel, docetaxel, arsenic trioxide, or tretinoin. Toxins includePseudomonas exotoxin (PE38), ricin A chain, diphtheria toxin, Besides PEand RT, Pokeweed antiviral protein (PAP), saporin and gelonin.Radionuclides for cancer therapy include Y-90, P-32, I-131, In-111,Sr-89, Re-186, Sm-153, and Sn-117m.

Agents or factors suitable for use in a combined therapy against anautoimmune disorder include anti-inflammatories and immune-modulatingcompositions. These include prednisone, methylprednisone, Venipred,Celestone, hydrocortisone, triamcinoclone, Aristonpan Intra-Articularinjection, Methapred, Rayos oral, betamethasone, and etanercept.

Agents or factors suitable for use in a combined therapy againstcardiovascular disease include beta blockers, anti-hypertensives,cardiotonics, anti-thrombotics, vasodilators, hormone antagonists,endothelin antagonists, cytokine inhibitors/blockers, calcium channelblockers, phosphodiesterase inhibitors and angiotensin type 2antagonists.

Agents or factors suitable for use in a combined therapy against ahypercoagulation disorder include anti-coagulants such as heparin andwarfarin.

Agents or factors suitable for use in a combined therapy against a viralinfections include Abacavir, Aciclovir, Acyclovir, Adefovir, Amantadine,Amprenavir, Ampligen, Arbidol, Atazanavir, Atripla, Boceprevirertet,Cidofovir, Combivir, Darunavir, Delavirdine, Didanosine, Docosanol,Edoxudine, Efavirenz, Emtricitabine, Enfuvirtide, Entecavir, Entryinhibitors, Famciclovir, Fomivirsen, Fosamprenavir, Foscarnet, Fosfonet,Ganciclovir, Ibacitabine, Imunovir, Idoxuridine, Imiquimod, Indinavir,Inosine, Integrase inhibitor, Interferon type III, Interferon type II,Interferon type I, Interferon, Lamivudine, Lopinavir, Loviride,Maraviroc, Moroxydine, Methisazone, Nelfinavir, Nevirapine, Nexavir,Nucleoside analogues, Oseltamivir, Peginterferon alfa-2a, Penciclovir,Peramivir, Pleconaril, Podophyllotoxin, Protease inhibitor, Raltegravir,Reverse transcriptase inhibitor, Ribavirin, Rimantadine, Ritonavir,Pyramidine, Saquinavir, Stavudine, Synergistic enhancer(antiretroviral), Tea tree oil, Telaprevir, Tenofovir, Tenofovirdisoproxil, Tipranavir, Trifluridine, Trizivir, Tromantadine, Truvada,Valaciclovir, Valganciclovir, Vicriviroc, Vidarabine, Viramidine,Zalcitabine, Zanamivir and Zidovudine.

The skilled artisan is directed to “Remington's Pharmaceutical Sciences”15th Edition, chapter 33, in particular pages 624-652. Some variation indosage will necessarily occur depending on the condition of the subjectbeing treated. The person responsible for administration will, in anyevent, determine the appropriate dose for the individual subject.Moreover, for human administration, preparations should meet sterility,pyrogenicity, general safety and purity standards as required by FDAOffice of Biologics standards.

It also should be pointed out that any of the foregoing therapies mayprove useful by themselves in treatment.

V. EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the disclosure. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the disclosure, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe disclosure.

Example 1

The affinities of Gla-domain proteins for cell membranes have beendetermined in vitro by using prepared phospholipid vesicles (Shah etal., 1998; Nelsestuen, 1999). How these in vitro values translate to anin vivo context, however, has not been fully elucidated. The interactionof FVII with TF, for example, underscores the fact that although the Gladomains of these proteins are very homologous, additional differences intheir cell membrane binding specificity and affinity may be mediatedthrough their EGF and/or Kringle domains. Unfortunately, theseinteractions cannot be recapitulated by studies based solely onphospholipid vesicles and may remain unidentified.

Therefore, inventors proposed making and testing the Gla+EGF/Kringledomains as well as the Gla domain alone from the following panel ofproteins: hS (high affinity binder), hZ (mid affinity binder), hPT (midaffinity-kringle containing), hFVII (low affinity-utilizes secondary“receptor” that is also up-regulated in cancer), and B0178 (hFVII withincreased phospholipid affinity). These proteins potentially havevarying in vivo binding characteristics that may be beneficial to theiruse as probes (and, if validated and selective, potentially astherapeutics) and that to date have gone unrecognized.

The general approach was to construct recombinant proteins and test themfor expression. Assays would then be developed to assess binding. Then,expression and purification methods would be optimized, followed byquality control of gamma-carboxylation.

FIG. 1 shows sequences from a variety of Gla domain proteins includingcarboxylation sites. FIG. 2 shows the expression of a variety ofdifferent Gla domain proteins that were engineered and transientlyexpressed in 293 cells. FIG. 3 shows a similar study in BHK21 cells.Given that one of the best expressing constructs was a Protein S+EGFconstruct, the signal sequence from Protein S was utilized withProthrombin Gla+Kringle and Protein Z+EGF. However, expression was onlyobserved intracellularly (FIG. 4).

Protein S Gla+EGF was selected for further study. The sequence is shownin FIG. 5. Protein was produced in BHK21 cells using RF286 medium. 600ml was harvested and concentrated 4×. Purification utilized three steps:

-   -   1. Ni-NTA column, 10 ml, fresh packed. The medium are loaded to        column and eluted with Imidazole gradient. All the fractions are        subject to Gla western blot to identify the His tagged Gla        protein S G+E.    -   2. Hitrap Q with CaCl₂ step elution. The Gla positive fractions        are pooled and subject to 1 ml Hitrap Q with 10 mM CaCl₂        elution.    -   3. Hitrap Q with CaCl₂ gradient (0-10 mM shadow gradient). The        step purified Gla proteins were applied to Q and eluted with        gradient CaCl₂ (up to 10 mM). A total of 0.9 mg of protein at a        95% purity level was produced. FIG. 6 shows the purification        fractions under both reducing and non-reducing conditions. FIGS.        7 and 8 show different FACs-based apoptosis assays. Both show        that the Protein S Gla+EGF construct is specific for cells        undergoing apoptosis just like Annexin V (FIG. 7), and that        Annexin V can compete off the Protein S Gla+EGF binding.

In summary, Protein S Gla+EGF was expressed and purified. Analysis onthe purified material suggested that it was highly gamma-carboxylated.FACs-based Apoptosis Assays demonstrated that Protein S G+E (11 Gla)could bind to “apoptotic” cells, and that this binding was to cells wasvia targeting of phosphatidylserine, as demonstrated by Annexin Vcompetition assays.

All of the compositions and/or methods disclosed and claimed herein canbe made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of thisdisclosure have been described in terms of preferred embodiments, itwill be apparent to those of skill in the art that variations may beapplied to the compositions and/or methods and in the steps or in thesequence of steps of the method described herein without departing fromthe concept, spirit and scope of the disclosure. More specifically, itwill be apparent that certain agents which are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of thedisclosure as defined by the appended claims.

VI. REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference:

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The invention claimed is:
 1. A method of targeting cell membranephosphatidylserine comprising: (a) providing an isolated polypeptide offrom 4.5 to 30 kD in size comprising a gamma-carboxyglutamic-acid (Gla)domain with 5-15 Gla residues, a protein S EGF domain and lacking aprotease or hormone-binding domain; and (b) contacting said peptide witha cell surface in vitro or ex vivo, wherein said polypeptide binds tophosphatidylserine on said cell membrane.
 2. The method of claim 1,wherein said cell membrane is a cardiac cell membrane, a neuronal cellmembrane, an endothelial cell membrane, a virus-infected cell membrane,an apoptotic cell membrane, a platelet membrane or a cancer cellmembrane.
 3. The method of claim 1, wherein said polypeptide furthercomprises a detectable label, wherein said detectable label comprises afluorescent label, a chemiluminescent label, a radio label, an enzyme, adye, or a ligand.
 4. The method of claim 1, wherein said polypeptidecomprises 300 residues or less.
 5. The method of claim 1, wherein saidpolypeptide comprises from 1-5 disulfide bonds.
 6. The method of claim1, wherein said polypeptide further comprises an antibody Fc region.